Recently alarming reports have been published, from which an impurification of the ground water, from which frequently drinking water is obtained without great expenditure for purification, by nitrates from fertilizers, by chlorinated solvents from dry cleaning operations, by herbicides (pesticides), insecticides and their decomposition products, emerges. In this process, the pesticide atrazine and its metabolites constitute the greatest problematical substance in drinking water.
Atrazine is a non-odiferous white powder having the chemical name 2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine and it has been used for approximately thirty years all over the world as a herbicide. It prevents a photosynthesis and thereby causes "weeds" rapidly to wither. Atrazine is used in particular as a weed killer in corn fields, and 90% of all the corn fields in the Federal Republic of Germany are treated with atrazine, so that atrazine heads the list of substances used, with approximately 1000 tonnes per annum. Apart from atrazine, other triazine herbicides are employed, the presence of which in the ground and surface waters has been proven. Table 1 provides a survey of the most important triazine herbicides.
TABLE 1 ______________________________________ Triazine-Herbicides ______________________________________ ##STR1## Ametryn: Atraton: R.sup.1 = SCH.sub.3, R.sup.2 = NHCH(CH.sub.3).sub. 2, R.sup.3 = NHC.sub.2 H.sub.5 R.sup.1 = OCH.sub.3, R.sup.2 = NHCH(CH.sub.3).sub.2, R.sup.3 = NHC.sub.2 H.sub.5 Atrazin: R.sup.1 = Cl, R.sup.2 = NHC.sub.2 H.sub.5, R.sup.3 = NHCH(CH.sub.3).sub.2 Aziprotryn: R.sup.1 = SCH.sub.3, R.sup.2 = NHCH(CH.sub.3).sub.2, R.sup.3 = N.sub.1 Desmetryn: R.sup.1 = SCH.sub.3, R.sup.2 = NHCH(CH.sub.3).sub.2, R.sup.3 = NHCH.sub.3 Dipropetryn: R.sup.1 = SC.sub.2 H.sub.5, R.sup.2 = R.sup.3 = NHCH(CH.sub.3).sub.2 Methoprotryn: R.sup.1 = SCH.sub.3, R.sup.2 = NHCH(CH.sub.3).sub.2, R.sup.3 = NHC.sub.3 H.sub.6OCH.sub.3 Prometryn: R.sup.1 = SCH.sub.3, R.sup.2 = R.sup.3 = NHC.sub.3 H.sub.7 Propazin: R.sup.1 = Cl, R.sup.2 = R.sup.3 = NHC.sub.3 H.sub.7 Simazin: R.sup.1 = Cl, R.sup.2 = R.sup.3 = NHC.sub.2 H.sub.5 Terbumeton: R.sup.1 = OCH.sub.3, R.sup.2, R.sup.3 wie Terbutryn Terbuthylazin: R.sup.1 = Cl, R.sup.2, R.sup.5 wie Terbutryn Terbutryn: R.sup.1 = SCH.sub.3, R.sup.2 = NHC(CH.sub.3).sub.3, R.sup.3 = NHC.sub. 2 H.sub.5 ______________________________________
For a long time, it was assumed that these triazine compounds are permanently decomposed after use and are bound with particles of soil, so that danger to the ground water is excluded. But it has been found that the compounds are very stable, and that the time taken for half of the active ingredient to be decomposed in the soil amounts to between two and five months. In sandy soil as well as in soils which have little loam and clay, the active ingredient is relatively easily washed out into the ground water, in which the decomposition takes still longer, so that residues of triazines may appear years later in the ground water.
The drinking water regulations and the "European Community Guidelines on the Quality of Water for Human Consumption" lay down limit values for the amounts which are still tolerable of these substances in drinking water. For individual substances the maximal limit amounts to 0.0001 mg/l (100 ng/l), as a whole, the sum of these substances may not exceed the concentration of 0.0005 mg/l (500 ng/l). For a precise analysis, the detection limit should be one or two orders of magnitude below this value. However, the great number of substances in question and the low limit values raise a great problem for chemical analysis. Physical-chemical methods of analysis (GC,GC-MS, HPLC) require large-scale enrichment processes for identification and for quantification of chemical substances and they are very costly and time-consuming. In addition they do not allow any statement concerning the toxicity of the compounds. Modern biochemical analytical processes are represented by immunological test processes, the so-called "immuno-assays", in which cell components are used as the test substances. Immunological analytical processes constitute highly-sensitive rapid tests in the sector of waters analysis, and they guarantee frequently fast, economical and effective environmental monitoring.
Immuno-assays are highly sensitive test systems for the quantitative detection of substances on the basis of the antigen-antibody reaction. The antibodies are firstly induced by the immunization of laboratory animals and are extracted from their sera or from lymphocytes producing antibodies, and are purified via affinity columns, which contain as the filler material agarose, for example. Antibodies represent one of the natural defensive systems of higher animal life. The defensive function is based on the fact that specific antibodies are induced on the basis of natural infection or of artificial infection by vaccination. A definite size of molecule is a prerequisite for the formation of antibodies. If a specific antibody formation is to be initiated against very small molecules, for example plant protection agents, these (haptenes) must be coupled before immunization with a high-molecular carrier molecule, such as e.g. a protein (haemocyanine, beef serum albumin, ovalbumin, thyroglobulin, polylysin et al.).
For the detection of chemical substances, the antigen-antibody bonding is employed, which takes place when adding a sample which contains the chemical substance as antigen to the antibody. In the classical immunoassay the antigens to be detected compete with radioactively marked antigens of the same specificity around the bonding positions on the antibody. More recently enzyme immunoassay (EIA) has increased in significance in comparison to radio immunoassay (RIA). In this process the radioactive antigen is replaced by an enzyme-antigen conjugate, which can then compete with the sample antigen for the free bonding positions of the antibody. In this competing EIA, a predetermined quantity of the enzyme marked antigen competes with the sample antigens for the bonding positions on antibodies, which for their part are bonded adsorptively or covalently to a substrate, for example a polystyrene surface. With a small concentration of sample antigens many enzyme tracers are bonded (high substrate conversion), while with a high concentration of sample antigens, on the other hand, only a few enzyme tracers are bonded (low substrate conversion). Therefore the amount of the bonded enzyme tracer is a function of the sample antigen concentration. After the separation of the non-bonded enzyme tracer parts by means of a washing step, it is possible to detect the share of the bonded enzyme marked antigen on the basis of the conversion of the substrate, and therefore to detect the antigen concentration in the sample. The measurement magnitude for the bonding of the enzyme tracer is the absorption of the converted quantity of substrate.
Using the technology described above, specific antibodies are induced against various pesticides and are developed in immuno-assays with the aim of rapid, highly-specific and therefore economical detection of this problematic substance. However, it is often the case that with such an immuno-assay clear identification and quantification is not possible because of cross-reactions. By a cross-reaction what is means is the phenomenon that antibodies recognize common functionalities in molecules of different structures and classify these molecules in one activity class but are unable to differentiate between the individual molecules. This effect is found very clearly in the case of small molecules (antigens), because these must be bonded on a carrier matrix (protein) and therefore only the side facing away from the carrier matrix, but not the entire molecule, acts as the recognition region. Molecules with similar structure to the side which is turned away can then often not be distinguished in immunoassays. In many cases, on the other hand, the effect of the cross-reaction is even desirable, vice-versa, because not only the antigen used specially for the production of an antibody, but also compounds having a similar chemical structure or with similar biological activity to that of the antigen used can be detected. By a test with the deliberate use of cross-reactions, different substances which belong to the same activity class can be detected. This is very valuable in the toxicity evaluation of previously unresearched compounds. Animal tests and bioassays can therefore be reduced to the minimum necessary, because the potential danger of ecotoxicologically previously unresearched compounds is already recognizable in the foreground due to the activity class classification mentioned above.